Biochemical screens for small molecules that correct protein misfolding currently require large amounts of well-behaved, pure protein. Such screens are time consuming, expensive, limited to soluble pre-folded substrates, and lack the capacity to access intermediate folding states that may be defective in protein folding disorders.
One example of such a screen is a screen for small molecules that corrects misfolding and subsequent degradation of CFTR proteins. One common mutation in cystic fibrosis patients is a deletion of the phenylalanine at amino acid position 508. This mutation is referred to in the art and herein as ΔF508 CFTR. F508 is located in the first nucleotide binding domain (NBD1) of CFTR. The ΔF508 mutation has detrimental effects on the folding efficiency of the NBD1 in cells, and on the thermodynamic stability of NBD1 in vitro. The efficiency of NBD1 folding is a limiting factor in CFTR trafficking, which is further compromised by the ΔF508 mutation.
An important goal in cystic fibrosis (CF) therapeutics, therefore, is to understand how the NBD1 domain acquires and maintains its folded state in the cellular environment and to devise pharmacological strategies to improve folding efficiency. Clearly, methods that allow more efficient study of protein folding and that identify compounds that improve the folding efficiency of the NBD1 domain and other misfolded proteins that cause disease are needed.
As described in Khushoo et al, Mol Cell 41, 682-692 (2011), the entirety of which is hereby incorporated herein by reference for all purposes, FRET-based methods can be used in cell free translation systems to define the NBD1 folding pathway and showed that cotranslational NBD1 folding begins with compaction of the N-terminal ATP-binding subdomain, followed by alpha-subdomain folding and lastly, formation of the central α/β-sheet core. Thus, the ability to monitor folding directly on the ribosome is desirable because it allows access to folding intermediates that exist only transiently during translation.
However, while cell free translation systems enable quantitative incorporation of fluorescent probes, the concentration of proteins obtained from stalled ribosomes is exceedingly low (e.g., approximately 1 nM). This poses significant challenges to identifying small molecules that might promote protein stability and/or folding efficiency.